Single domain antibodies targeting CD1d

ABSTRACT

The invention relates to compounds, in particular polypeptides that specifically bind to the non-classical MHC protein CD1d and modulate CD1d-mediated biological functions. The invention in particular relates to such compounds and polypeptides comprising or consisting of at least one single domain antibody, and wherein at least one single domain antibody specifically binds to CD1d. Also provided is for methods and use employing such compounds, polypeptides and/or single-domain antibodies.

The Sequence Listing in ASCII text file format of 40,626 bytes in size,created on Nov. 2, 2017, with the file name“2017-12-11SequenceListing_VANDERVLIET1,” filed in the U.S. Patent andTrademark Office on Dec. 11, 2017, is hereby incorporated herein byreference.

FIELD OF INVENTION

The present invention generally relates to the field of immunology, morein particular to the field of single-domain antibodies which bind tohuman CD1d, including antibodies that modify CD1d-mediated biologicalfunctions such as activation of CD1d-restricted T cells, including thenatural killer T (NKT) cells, and modulation of the function of cellsexpressing CD1d. Provided are, for example, compounds which comprise atleast one single-domain antibody which binds to CD1d, use of suchcompounds comprising at least one single-domain antibody and(pharmaceutical) compositions comprising such compounds.

BACKGROUND ART

CD1d is a member of the CD1 (cluster of differentiation 1) family ofglycoproteins (including CD1a, CD1b, CD1c, CD1d and CD1e) expressed onthe surface of various human cells, including antigen presenting cells(APC). In human CD1d is encoded by CD1D, also known as R3G1. APCdisplaying CD1d include Langerhans cells, (activated) B-cells, dendriticcells (e.g. in lymph nodes), and (activated) blood monocytes. CD1d isalso expressed by various other cell types, for example in liver,pancreas, skin, kidney, uterus, conjunctiva, epididymis, thymus andtonsil (see, for example, Canchis et al. (1992) Immunology 80:561-565).

Cells that are activated/stimulated via CD1d include the Natural KillerT-cells (NKT cells). NKT cells are a heterogeneous group of T cells thatshare properties of both T cells and natural killer cells. NKT cells area subset of T cells that express an alpha/beta T-cell receptor (TCR), aswell a variety of molecular markers that are typically associated withNKT cells.

Type 1 or invariant NKT cells is the best-known group of NKT cells anddiffers from conventional αβ T cells in that their T-cell receptors arefar more limited in diversity (‘invariant’). The NKT cells, includingthese invariant and other CD1d-restricted T cells (type 2 NKT),recognize (self or foreign) lipids and glycolipids presented by CD1dmolecules present on APC. The interaction between (lipid-presenting)CD1d and TCR triggers the release of cytokines including Th1- orTh2-like cytokines, such as interferon-gamma, tumor necrosisfactor-alpha, and interleukins like IL-4, IL-5 and IL-13.

Different lipids have been shown to bind CD1d molecules, includingmycolic acids, diacylglycerols, and sphingolipids. Analpha-galactosylceramide, KRN7000, is the best studied ligand of thelipid-binding CD1d in NKT cell activation in vitro and in vivo. Otherligands comprise isoglobotrihexosylceramide, (microbial-derived)glycuronosylceramides, alpha-C-galactosylceramides, threitol ceramide,and a variety of (human and non-human) glycolipids such aslysophophatidylcholine and lysosphingomyelin (see, for example, Fox etal (2009) PLOS Biology 7:10:e1000228).

Important roles of NKT cells have now been demonstrated in theregulation of autoimmune, allergic, antimicrobial, and antitumor immuneresponses (van der Vliet et al. (2004) Clinical Immunology 112(1):8-23). Physiologically, the NKT-cells can augment or inhibit immuneresponses, including antitumor, autoimmune, and anti-pathogen responses,through a variety of mechanisms depending on context (Yue et al. (2010)The Journal of Immunology 184: 268-276), including induction of celldeath in multiple myeloma cells. Conditions in which NKT-cells may beinvolved include autoimmune or inflammatory diseases, includingmyasthenia gravis, psoriasis, ulcerative colitis, primary biliarycirrhosis, colitis, autoimmune hepatitis, atherosclerosis, and asthma.In addition to cytokine release, NKT cell effector functions whichresult in cell lysis, such as perforin release and granzyme release andcell death, may also be relevant in conditions in which NKT cells areimplicated, such as in cancer. Modulation of CD1d-mediated effects istherefore of potential therapeutic benefit.

There is an ongoing need for compounds that can bind and/or interactwith CD1d as specific as possible, i.e. while minimally or not bindingto other family members of the CD1-family, both in vitro and in vivo. Inparticular there is need for such compounds that bind and/or modulate(activate or inhibit) biological functions that involve CD1d such as,but not limited to, NKT-cell activation. Such compounds may, forexample, show benefit in the various diseases in which CD1d-mediatedfunctions play a role.

SUMMARY OF THE INVENTION

The present invention provides a compound comprising at least onesingle-domain antibody. The single-domain antibody binds to human CD1d.The single-domain antibody that binds to human CD1d comprises a CDR1,CDR3 and CDR3 region with an amino acid sequence as disclosed herein,and conservative sequence variants thereof.

Preferably the single-domain antibody has a CDR1, CDR2 and CDR3 regionin the combination as disclosed herein, for example as shown in Tabel1.

Even more preferably, the single domain antibody has an amino acidsequence selected from the group of SEQ ID NO: 1-SEQ ID NO: 21.

The compound according to the invention may be any kind of compound, forexample a complex, as long as the single-domain antibody that binds tohuman CD1d is comprised in the compound. Preferably the compound is apolypeptide. In certain embodiments the compound may consist of only thesingle-domain antibody that binds to human CD1d. In other embodimentsthe compounds consists of the single-domain antibody that binds to humanCD1d and a label. In even further embodiments the compound may comprisethe single-domain antibody that binds to human CD1d linked to apharmaceutical active agent and/or other antibodies.

Also provided is use of the compound according to the invention inmedical treatment and/or as a diagnostic agent.

Also provided is a pharmaceutical composition that comprises a compoundas disclosed herein and nucleotide sequences and host cells comprisingsuch nucleotide sequences that encode for the compounds according to theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1: CD1d-specificity of individual selected nanobodies.Flow-cytometry was used to detect binding of isotype control mAb(IgG2b), anti-CD1d 51.1 mAb, R2 negative control VHH, and individualCD1d-specific VHH. Data demonstrate binding to CD1a, CD1b, CD1c, andCD1d transfected tumor cell lines (n=3).

FIG. 2: Induction of moDC maturation and cytokine production byCD1d-specific nanobodies. Immature moDC were cultured with CD1d-specificnanobodies. After 24 hours, supernatants were harvested for detection ofcytokine production (ELISA). After 72 h, moDC were analyzed for cellsurface expression of the maturation marker CD83 using flow cytometry.NC=negative control, PC=positive control, IgG2b=isotype control mAb,51.1=anti-CD1d 51.1 mAb, LPS=lipopolysaccharide,LPS-PMB=lipo-polysaccharide with polymyxin B. Data represent mean+SEM,n=3.

FIG. 3: Inhibition of α-GalCer induced iNKT cell activation.CD1d-transfected HeLa cells were pulsed overnight with vehicle control(veh) or α-GalCer (all other conditions). After washing, vehicle orα-GalCer pulsed HeLa-CD1d were cultured for 2 hours with IgG2b isotypecontrol mAb, anti-CD1d 51.1 mAb, negative control VHH R2, or aneutralizing anti-CD1d VHH (VHH 24 (18-29c)) after which time iNKT cellswere added. After 24 hr iNKT cell activation (CD25 expression) wasdetermined using flow cytometry. Data indicate mean+SEM of 3experiments. Superior neutralization of iNKT cell activation byanti-CD1d VHH.

FIG. 4: Induction of iNKT cell activation. CD1d-transfected C1R cellswere pulsed overnight with vehicle control (veh) or α-GalCer asindicated. After washing, vehicle or α-GalCer pulsed C1R-CD1d werecultured for 2 hours with or without a specific anti-CD1d VHH afterwhich iNKT cells were added. After 24 hr iNKT cell activation (CD25expression) was determined using flow cytometry. Representative flowcytometric dotplots demonstrating activation of iNKT cells by α-GalCer,but more strikingly after co-culture with the anti-CD1d VHH (VHH12(18-14b)). Data are representative from multiple experiments withmultiple CD1d-expressing tumor cell lines.

FIG. 5: Induction of annexin V binding by anti-CD1d nanobodies. C1Rcells, CD1d-transfected C1R cells (left panel) and MM.1s cells andCD1d-transfected MM.1s cells (right panel) were cultured for 24 hourswith IgG2b isotype control mAb, anti-CD1d 51.1 mAb, negative control VHHR2, or a CD1d-specific VHH (VHH19 (19-23G)). Percentage of target cellsbinding annexin V, which is suggestive of early apoptosis, was thendetermined by flow-cytometry. Data indicate mean+SEM of 3 experiments.

FIG. 6: Induction of iNKT cell activation using platebound β2m-humanCD1d (±vehicle, α-GalCer and/or anti-CD1d VHH). 96-well plates werecoated with a bispecific construct consisting of anti-EGFR VHH fused toβ2m-hCD1d (loaded with either vehicle control or α-GalCer). Coatedplates were cultured for 2 hours in the presence or absence of ananti-CD1d VHH (VHH12) after which iNKT cells were added. After 24 hoursiNKT cell activation (CD25 expression) was determined usingflowcytometry. Representative flowcytometric dotplots demonstratingslight activation of iNKT cells by α-GalCer-loaded β2m-hCD1d, but robustactivation after co-culture of α-GalCer-loaded β2m-hCD1d with theanti-CD1d VHH (VHH12 (18-14b)).

FIG. 7: Dose dependent inhibition of CD1d-α-GalCer mediated iNKT cellactivation. iNKT CD25 expression, IFN-γ and TNF-α production weredetermined after a 24 h co-culture of iNKT with CD1d-transfected HeLacells pulsed with vehicle control (vehicle) or α-GalCer (all otherconditions) and medium (vehicle and α-GC), anti-CD1d mAb 51.1 (10μg/ml), control VHH (500 nM) or anti-CD1d VHH (VHH24; 500 nM). Graphicalrepresentation showing CD25 expression on iNKT cells (a). Concentrationdependent effect of anti-CD1d VHH (● symbols) and a controlnon-inhibitory but CD1d-specific VHH (▴ symbols) on IFN-γ and TNF-αproduction. ♦ indicate the vehicle loaded control condition (b).Mean+SD, n=3, **p<0.05, **p<0.01, ****p<0.0001. The tested VHH is VHH24.

FIG. 8: Dose dependent iNKT cell activation by anti-CD1d VHH12. CCRF-CEM(T-ALL, CD1d positive; n=4) and CD1d-transfected MM.1s (multiplemyeloma; n=3) cells were pulsed with vehicle control or αGC, incubatedwith anti-CD1d VHH and controls and co-cultured with iNKT for 24 h afterwhich iNKT CD25 expression was determined. *p<0.05, **p<0.01,***p<0.001, ****p<0.0001 (compare to FIG. 4).

FIG. 9: Induction of iNKT cell degranulation (left) and cytotoxicityagainst CD1d+ tumor cells line (right). CCRF-CEM cells (CD1d-positive)were pulsed with vehicle control or αGC, incubated with anti-CD1d VHHand controls and co-cultured with iNKT (E:T ratio of 1:2) for theindicated time 6, 12 or 18 h) and stained with CD107a (t=4 h) or annexinV and 7-AAD for flow cytometry. N=5; *p<0.05; ***p<0.001. The anti-CD1dVHH shown is VHH12.

FIG. 10: Induction of iNKT cell cytotoxicity against CD1d+ primarymultiple myeloma cells. Thawed primary bone marrow samples from MMpatients were pulsed with vehicle control or αGC or incubated withanti-CD1d VHH and controls and then co-cultured with iNKT for theindicated time (8 and 16 h) after which the percentage of surviving MMcells was determined. The anti-CD1d VHH shown is VHH12.

FIG. 11: Induction of iNKT cell cytokine production by anti-CD1d VHH12.For detection of cytokine production HeLa-CD1d cells were pulsed withvehicle control, OCH (a sphingosine truncated analog ofalpha-galactosylceramide (alpha-GC); glycolipid reported to induceTh2-cytokine production in iNKT cells) or αGC, incubated with anti-CD1dVHH and controls and co-cultured with iNKT for 24 h after whichsupernatants were analyzed (by Cytometric Bead Assay; CBA). N=4;*p<0.05; ****p<0.0001.

DETAILED DESCRIPTION OF THE INVENTION Definitions

In the following description and examples a number of terms are used. Inorder to provide a clear and consistent understanding of thespecifications and claims, including the scope to be given to suchterms, the following definitions are provided. Unless otherwise definedherein, all technical and scientific terms used have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. The disclosures of all publications, patentapplications, patents and other references are incorporated herein intheir entirety by reference.

Methods of carrying out the conventional techniques used in methods ofthe invention will be evident to the skilled worker. The practice ofconventional techniques in molecular biology, biochemistry,computational chemistry, cell culture, recombinant DNA, bioinformatics,genomics, sequencing and related fields are well-known to those of skillin the art and are discussed in handbooks.

In this document and in its claims, the verb “to comprise” and itsconjugations is used in its non-limiting sense to mean that itemsfollowing the word are included, but items not specifically mentionedare not excluded. It encompasses the verbs “consisting essentially of”as well as “consisting of”.

As used herein, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. For example, amethod for using “a” compound, includes using a plurality of thiscompound (e.g. 10s, 100s, 1000s, 10s of thousands, 100s of thousands,millions, or more molecules).

With the term “aligning” and “alignment” is meant the comparison oramino acid sequences of two or more molecules/compounds based on thepresence of short or long stretches of identical or similar amino acids.Several methods for alignment of amino acid sequences are known in theart, as will be further explained below.

“Sequence identity” is a measure of the identity of nucleotide sequencesor amino acid sequences. In general, the sequences are aligned so thatthe highest order match is obtained. “Identity” per se has anart-recognized meaning and can be calculated using published techniques.Methods commonly employed to determine identity or similarity betweentwo sequences include, but are not limited to, those disclosed in GUIDETO HUGE COMPUTERS, Martin J. Bishop, ed., Academic Press, San Diego,1994, and Carillo, H., and Lipton, D., SIAM J. Applied Math (1988)48:1073. Methods to determine identity and similarity may be codified incomputer programs. Preferred computer program methods to determineidentity and similarity between two sequences include, but are notlimited to, GCS program package (Devereux, J., et al., Nucleic AcidsResearch (1984) 12(1):387), BLASTP, BLASTN, FASTA (Atschul, S. F. etal., J. Molec. Biol. (1990) 215:403). Sequence identity as disclosedherein was determined by calculating) the percentage of amino acids thatare similar (number of amino acids similar to reference sequence dividedby total number of amino acids in the reference sequence), essentiallyas outlined in the paragraph below.

As an illustration, by an amino acid sequence with at least, forexample, 70% “sequence identity” to a reference amino acid sequence ofSEQ ID NO: 1 it is intended that the amino acid sequence is identical tothe reference sequence except that the polypeptide sequence may includeup to 3 amino acid alterations per each of the 10 amino acids of thereference amino acid of SEQ ID NO: 1. Hence, the percentage of identityof an amino acid sequence to a reference amino acid sequence is to becalculated over the full length of the reference amino acid sequence. Inother words, to obtain an amino acid sequence comprising at least 70%identical to a reference amino acid sequence, up to 30% of the aminoacid residues in the reference sequence may be deleted or substitutedwith another amino acid, or a number of amino acids up to 30% of thetotal amino acid residues in the reference sequence may be inserted intothe reference sequence. These alterations of the reference sequence mayoccur at the amino- or carboxy-terminal positions of the reference aminoacid sequence or anywhere between those terminal positions, interspersedeither individually among residues in the reference sequence or in oneor more contiguous groups within the reference sequence.

The terms “amino acid sequence” or “protein” or “peptide” refer tomolecules consisting of a chain of amino acids, without reference to aspecific mode of action, size, 3 dimensional structure or origin. A“fragment” or “portion” of thereof may thus still be referred to as an“amino acid sequence” or “protein” or “peptide”. An “isolated amino acidsequence” is used to refer to an amino acid chain with a particularsequence and which is no longer in its original natural environment, forexample in vitro or in a recombinant bacterial or human host cell.

Each immunoglobulin molecule has a variable domain. The variable domainof immunoglobulin molecules is subdivided into hypervariable (HV) andframework (FR) regions. HV regions have a high ratio of different aminoacids in a given position, relative to the most common amino acid inthat position. The hypervariability regions are referred to ascomplementarity determining regions (CDR). Immunoglobulin molecules havethree complementarity determining regions (CDR1, CDR2 and CDR3). Fourframework regions, with much less variable amino acids sequences,separate the CDR regions. The CDR regions can direct binding to theantigen, such as CD1d.

Description

The present invention generally relates to compounds comprisingsingle-domain antibodies which bind to human CD1d. The present inventorshave found single-domain antibodies and antigen-binding portions thereofwhich bind to human CD1d.

In a first aspect there is provided for a compound comprising at leastone single-domain antibody which binds to human CD1d, wherein thesingle-domain antibody comprises complementarity determining regionsCDR1, CDR2 and CDR3, wherein CDR1 comprises an amino acid sequence thathas at least 60% sequence identity with SEQ ID NO: 22 and CDR2 comprisesan amino acid sequence that has at least 60% sequence identity with SEQID NO: 43.

As disclosed before, CD1d (Entrez Gene ID 912; NCBI Reference Sequence:NP_001757; Balk et al. (1989) Proc Natl Acad Sci USA 86:252-256) isexpressed in a variety of cells including B-cells in chronic lymphocyticleukemia patients, hepatocytes, dendritic cells, and tumor cells and thesingle-domain antibodies disclosed herein can be used for binding toCD1d, for example, but not restricted to binding to CD1d on any of thesecells or for binding to CD1d on other cells expressing CD1d, or forbinding to CD1d molecules that are not bound to cells, and that areeither not bound to anything, or are for example linked to or associatedwith carriers, polymers or other proteins.

The compound comprising the single-domain antibody which binds humanCD1d can be any kind of compound or complex as long as it comprises asingle-domain antibody which binds to CD1d. Preferably, the compoundaccording to the invention can bind human CD1d due to the presence ofthe single-domain antibody which binds human CD1d.

The compound according to the invention may further comprise otherfunction or non-functional groups. For example, the single-domainantibody of the current invention may be linked to a nanoparticle, aliposome, a virus, a label, another antibody or protein structure (e.g.a receptor) or may be fused to an antigen, peptide, a drug, a marker, ornucleic acid. For example, the compound may also comprise a magneticbead, allowing the isolation of CD1d expressing cells.

The CD1d single-domain antibody may be linked via the carboxyl or aminoterminus of the antibody, or may be linked at a site other than thecarboxyl or amino termini. The attachment to the CD1d single-domainantibody may be direct, i.e., without any intermediate sequence, orthrough a linker amino acid sequence, a linker molecule, or a chemicalbond. For example, the coupling may be of a physical and/or chemicaltype.

In one embodiment, the compound is a bi-specific antibody or amulti-specific antibody. In one embodiment, the compound is a bivalentantibody or a multivalent antibody. Bivalency or multi-valency can allowantibodies to bind to multimeric antigen with great avidity;bi-specificity or multi-specificity can allow the cross-linking of twoantigens.

The compound comprises at least one single-domain antibody which bindsto human CD1d, wherein the single-domain antibody comprisescomplementarity determining regions CDR1, CDR2 and CDR3, wherein CDR1comprises an amino acid sequence that has at least 60% sequence identitywith SEQ ID NO: 22 and CDR2 comprises an amino acid sequence that has atleast 60% sequence identity with SEQ ID NO: 43.

Single domain antibodies (sdAb, also called Nanobody by Ablynx, thedeveloper, or VHH) are well known to the skilled person. Single domainantibodies are antibodies whose complementarity determining regions arepart of a single domain polypeptide. Single domain antibodies thuscomprise a single complementarity determining region (CDR) 1 (CDR1), asingle CDR2 and a single CDR3. Examples of single domain antibodies areheavy chain only antibodies, antibodies that naturally do not compriselight chains, single domain antibodies derived from conventionalantibodies, and engineered antibodies.

Single domain antibodies may be derived from any species includingmouse, human, camel, llama, goat, rabbit, and bovine. For example,naturally occurring VHH molecules can be derived from antibodies raisedin Camelidae species, for example in camel, dromedary, alpaca andguanaco.

Like a whole antibody, a single domain antibody is able to bindselectively to a specific antigen. Single domain antibodies may containonly the variable domain of an immunoglobulin chain having CDR1, CDR2and CDR3 and framework regions. With a molecular weight of only about12-15 kDa, nanobodies are much smaller than common antibodies (150-160kDa) which are composed of two heavy chains and two light chains.

CDR1, CDR2 and CDR3 sequences may be exchanged between species. Forexample, from a llama immunoglobulin molecule, CDR sequences may beselected and exchanged with CDR sequences in a human immunoglobulinmolecule, to obtain a human immunoglobulin molecule having thespecificity that is derived from the llama CDR sequences. This may beadvantageous as a human sequence may be less immunogenic to humans ascompared to the original llama framework sequence. Such an exchange ofCDR sequences is known as humanization.

Hence, the immunoglobulin molecules according to the invention may havehuman derived immunoglobulin sequences or llama derived immunoglobulinsequences and have the CDR1, CDR2 and CDR3 sequences replaced with theCDR sequences according to the invention in order to provide for humanCD1d binding. In other words, the compound according to the inventionmay comprise a humanized single-domain antibody with CDRs as disclosedherein. For example, a single domain antibody may have human frameworksequences and CDR regions as disclosed herein.

The single-domain antibody that is comprised in the compound accordingto the invention comprises complementarity determining regions CDR1,CDR2 and CDR3, wherein CDR1 comprises an amino acid sequence that has atleast 60% sequence identity with SEQ ID NO: 22 and CDR2 comprises anamino acid sequence that has at least 60% sequence identity with SEQ IDNO: 43.

The sequence of SEQ ID NO:22 correspond with the sequence of CDR1 of thesingle-domain antibody denoted as 17-1E in Table 1 herein. For thepurpose of the current invention the single-domain antibody 17-1E mayalso be referred to as VHH number 1. The sequence of the single-domainantibody 17-1E is shown as SEQ ID NO:1 and comprises, in addition to thesequences of the CDR1, CDR2 and CDR3 as shown in Table 1 also theframework sequences.

The sequence with SEQ ID NO:43 correspond with the sequence of CDR2 ofthe single-domain antibody denoted as 17-1E in Table 1 herein.

For all single-domain antibodies described herein and, for example, aslisted in Table 1, the region before CDR1 may be referred to asframework region (FW) 1, the region between CDR1 and CDR2 may bereferred to as FW2, the region between CDR2 and CDR3 may be referred toas FW3, and the region after CDR3 may be referred to as FW4. Therespective individual framework regions FW1, FW2, FW3 or FW4 can beeasily established based on the sequences of the CDR1, CDR2 and CDR3 andthe whole single-domain antibody, and are therefore disclosed as such.

It was surprisingly found that a variety of single-domain antibodiescould be obtained that share a high amino acid sequence identity withrespect to the CDR1 and CDR2 of the various single-domain antibodies.The CDR1, CDR2 and CDR3 sequences of the single-domain antibodies foundare listed in Table 1. For example, the single-domain antibody denotedas 19-23G in Table 1 has VHH number 19, and has a combination of a CDR1with a sequence that corresponds with SEQ ID NO:37, a CDR2 with asequence that corresponds with SEQ ID NO: 58 and a CDR3 with a sequencethat corresponds with SEQ ID NO: 79. The whole sequence, includingframework regions of this VHH is SEQ ID NO: 16.

However, according to the invention the single-domain antibody maycomprise any combination of a CDR1, CDR2 and CDR3 as long as CDR1 showsat least 60% sequence identity with SEQ ID NO: 22 and CDR2 comprises anamino acid sequence that has at least 60% sequence identity with SEQ IDNO: 43. For example, also contemplated is that the single-domainantibody comprises a CDR1 as shown in Table 1 of a first VHH (forexample VHH nr 10) and a CDR2 as shown in Table 1 of a second VHH (forexample VHH nr 20).

In other words, it will be appreciated that, based on the presentdisclosure, the skilled person can, without undue burden, providecompounds according to the invention, comprising at least onesingle-domain antibody which binds to human CD1d, wherein the CDR1 ofthe single-domain antibody comprises an amino acid sequence that has atleast 60% sequence identity with SEQ ID NO: 22 and the CDR2 of thesingle-domain antibody comprises an amino acid sequence that has atleast 60% sequence identity with SEQ ID NO: 43. For example, based onthe various CDR1s and CDR2 shown in Table 1.

In a preferred embodiment CDR1 over its entire length shows at least 60%sequence identity with SEQ ID NO: 22. In a preferred embodiment CDR2over its entire length shows at least 60% sequence identity with SEQ IDNO: 43. Preferable, CDR1 over its entire length shows at least 60%sequence identity with SEQ ID NO: 22 and CDR2 over its entire lengthshows at least 60% sequence identity with SEQ ID NO: 43. Preferably CDR1and/or CDR2 shows at least 65%, 70%, 75%, 80%, 90%, 95%, 97%, 99%identity with respectively SEQ ID NO:22 and/or SEQ ID NO: 43.

Also provided is a compound comprising at least one single-domainantibody which binds to human CD1d, wherein the single-domain antibodycomprises complementarity determining regions CDR1, CDR2 and CDR3 andwherein CDR1 comprises an amino acid sequence that has at least 90%sequence identity with SEQ ID NO: 22, CDR2 comprises an amino acidsequence that has at least 80% sequence identity with SEQ ID NO: 43, andCDR3 comprises an amino acid sequence that has at least 70% sequenceidentity with SEQ ID NO: 64; or wherein CDR1 comprises an amino acidsequence selected from the group consisting of SEQ ID NO: 33 and SEQ IDNO: 42, CDR2 comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO: 54 and SEQ ID NO: 63 and CDR3 comprises anamino acid sequence selected from the group consisting of SEQ ID NO: 75and SEQ ID NO: 84.

It was found that within the provided single-domain antibodies that canbe comprised in the compound according to the present invention, a groupis present that displays for the CDR1 an amino acid sequence that has atleast 90% sequence identity with SEQ ID NO: 22, for the CDR2 an aminoacid sequence that has at least 80% sequence identity with SEQ ID NO: 43and for the CDR3 an amino acid sequence that has at least 70% sequenceidentity with SEQ ID NO: 64. These single-domain antibodies share a highdegree of identity with respect to the respective complementaritydetermining regions. In a preferred embodiment CDR1 over its entirelength shows at least 90% sequence identity with SEQ ID NO: 22. In apreferred embodiment CDR2 over its entire length shows at least 80%sequence identity with SEQ ID NO: 43. In a preferred embodiment CDR3over its entire length shows at least 70% sequence identity with SEQ IDNO: 64. Preferable, CDR1 over its entire length shows at least 90%sequence identity with SEQ ID NO: 22, CDR2 over its entire length showsat least 80% sequence identity with SEQ ID NO: 43 and CDR3 over itsentire length shows at least 70% sequence identity with SEQ ID NO: 64.Preferably CDR1 shows at least 90%, 92%, 95%, 97%, 99% identity with SEQID NO:22, CDR2 shows at least 80%, 82%, 85%, 90%, 92%, 95%, 97%, 99%identity with SEQ ID NO: 43 and CDR3 shows at least 70%, 72%, 75%, 78%,80%, 82%, 85%, 90%, 92%, 95%, 97%, 99% identify with SEQ ID NO: 64.

According to the invention, in a preferred embodiment, the single-domainantibody may comprise any combination of a CDR1, CDR2 and CDR3 as longas CDR1 shows at least 90% sequence identity with SEQ ID NO: 22 and CDR2comprises an amino acid sequence that has at least 80% sequence identitywith SEQ ID NO: 43 and CDR3 comprises an amino acid sequence that has atleast 70% identity with SEQ ID NO:64. For example, also contemplated isthat the single-domain antibody comprises a CDR1 as shown in Table 1 ofa first VHH (for example VHH nr 10; SEQ ID NO: 31) and a CDR2 as shownin Table 1 of a second VHH (for example VHH nr 20; SEQ ID NO: 59), and aCDR3 as shown in Table 1 of the first or second VHH or of a third VHH(for example VHH nr 21; SEQ ID NO: 81).

In other words, it will be appreciated that, based on the presentdisclosure, in a preferred embodiment, the skilled person can, withoutundue burden, provide compounds according to the invention, comprisingat least one single-domain antibody which bind to human CD1d bycombining different CDR1, CDR2 and CDR3's, wherein the CDR1 of thesingle-domain antibody comprises an amino acid sequence that has atleast 90% sequence identity with SEQ ID NO: 22 and the CDR2 of thesingle-domain antibody comprises an amino acid sequence that has atleast 80% sequence identity with SEQ ID NO: 43 and the CDR3 of thesingle-domain antibody comprises an amino acid sequence that has atleast 70% sequence identity with SEQ ID NO: 64. For example, based onthe various CDR1, CDR2 and CDR3 shown in Table 1.

In another preferred embodiment, there is provided a compound comprisingat least one single-domain antibody which binds to human CD1d, whereinthe single-domain antibody comprises complementarity determining regionsCDR1, CDR2 and CDR3 wherein CDR1 comprises an amino acid sequenceselected from the group consisting of SEQ ID NO: 33 and SEQ ID NO: 42,CDR2 comprises an amino acid sequence selected from the group consistingof SEQ ID NO: 54 and SEQ ID NO: 63 and CDR3 comprises an amino acidsequence selected from the group consisting of SEQ ID NO: 75 and SEQ IDNO: 84.

Preferably, there is provided for compound comprising at least onesingle-domain antibody which binds to human CD1d as disclosed herein,wherein the single-domain antibody has complementarity determiningregions CDR1, CDR2 and CDR3 as listed in combination in Table 1, orconservative sequence variants thereof.

Although it will be appreciated that the skilled person will be able toprovide for various single-domain antibodies based on the various CDR1,CDR2, CDR3 as disclosed herein, as well as the other sequences provided(including the various framework sequences and the full-length sequenceof the single-domain antibodies), preferably the single-domain antibodyhas a CDR1, CDR2 and a CDR3 as shown in combination in Table 1, andconservative sequence variants thereof. In other words, a compoundaccording to the invention comprises a single-domain antibody wherein,preferably, the CDR1 and the CDR2 and the CDR3 are of one and the sameVHH as shown in Table 1. For example, the single-domain antibody has theCDR1, CDR2 and CDR3 of the same VHH as shown in Table 1, for example ofVHH1, VHH2, VHH3 . . . VHH14, VHH18, VHH 19 . . . VHH24. It was foundthat in particular CDR1, CDR2 and CDR3 as shown in combination (i.e.from the same VHH) show beneficial CD1d binding. As will be appreciatedby the skilled person, also included are conservative sequence variantsof the CDR1, CDR2 and CDR3 combinations as disclosed in Table 1.

Indeed in determining the degree of sequence identity between two aminoacid sequences or in establishing the CDR1, CDR2 and CDR3 combination inthe single-domain antibody, the skilled person may take into accountso-called “conservative” amino acid substitutions, which can generallybe described as amino acid substitutions in which an amino acid residueis replaced with another amino acid residue of similar chemicalstructure and which has little or essentially no influence on thefunction, activity or other biological properties of the polypeptide.Such conservative amino acid substitutions are well known in the art,for example from WO 04/037999, WO 00/46383, WO 01/09300 and WO04/037999. Conservative substitutions preferably are substitutions inwhich one amino acid within the following groups (a)-(e): An amino acidresidue is substituted by another amino acid residue within the samegroup (a)-(e): (a) small aliphatic, nonpolar or slightly polar residues:Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues andtheir (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positivelycharged residues: His, Arg and Lys; (d) large aliphatic, nonpolarresidues: Met, Leu, Ile, Val and Cys; and (e) aromatic residues: Phe,Tyr and Trp.

Preferred examples of conservative substitutions are as follows: Alainto Gly or into Ser; Arg into Lys; Asn into Gln or into His; Asp intoGlu; Cys into Ser, Gln into Asn; Glu into Asp; Gly into Ala or into Pro;His into Asn or into Gln; Ile into Leu or into Val; Leu into Ile or intoVal; Lys into Arg, into Gln or into Glu; Met into Leu, into Tyr or intoIle; Phe into Met, into Leu or into Tyr, Ser into Thr; Thr into Ser, Trpinto Tyr; Tyr into Trp; and/or Phe into Val, into Ile or into Leu.

Preferably, the single-domain antibody has complementarity determiningregions CDR1, CDR2 and CDR3 as listed in combination in Table 1,including conservative sequence variants thereof. More preferably, thesingle-domain antibody has complementarity determining regions CDR1,CDR2 and CDR3 as listed in combination in Table 1.

Also provided is a compound as disclosed herein and above wherein thecompound comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO: 1-SEQ ID NO: 21, or conservative sequencevariants thereof.

In other words, preferably, the single-domain antibody comprised in thecompound according to the invention comprises or consists of an aminoacid sequence selected from the group consisting of SEQ ID NO: 1-SEQ IDNO: 21, or, as explained above, conservative sequence variants thereof.These sequences 1-22 represent single-domain antibodies with the CDR1,CDR2 and CDR3 as shown in combination in Table 1, including theframework regions. These single-domain antibodies and there CD1d bindingproperties as described in detail in the Examples disclosed herein. SEQID NO:1 corresponds with VHH nr 1; SEQ ID NO:2 corresponds with VHH nr2; SEQ ID NO: 3 corresponds with VHH nr 3; SEQ ID NO:4 corresponds withVHH nr 4; SEQ ID NO:5 corresponds with VHH nr 5; SEQ ID NO: 6corresponds with VHH nr 6; SEQ ID NO:7 corresponds with VHH nr 7; SEQ IDNO:8 corresponds with VHH nr 8; SEQ ID NO: 9 corresponds with VHH nr 9;SEQ ID NO:10 corresponds with VHH nr 10; SEQ ID NO:11 corresponds withVHH nr 11; SEQ ID NO: 12 corresponds with VHH nr 12; SEQ ID NO:13corresponds with VHH nr 13; SEQ ID NO:14 corresponds with VHH nr 14; SEQID NO: 15 corresponds with VHH nr 18; SEQ ID NO:16 corresponds with VHHnr 19; SEQ ID NO: 17 corresponds with VHH nr 20; SEQ ID NO:18corresponds with VHH nr 21; SEQ ID NO:19 corresponds with VHH nr 22; SEQID NO: 20 corresponds with VHH nr 23; and SEQ ID NO:21 corresponds withVHH nr 24 as shown in Table 1.

Also provided are single-domain antibodies comprised in the compoundaccording to the invention and that has at least 70%, 80%, 85%, 90%,92%, 95%, 96%, 97%, 98% or at least 99% amino acid sequence identitywith a sequence selected from the group consisting of SEQ ID N: 1-21,over its entire length (and as applicable for all sequence disclosedherein).

Although the compound according to the invention may be any kind ofcompound comprising a single-domain antibody that binds CD1d, in apreferred embodiment the compound is a polypeptide to which apharmaceutical active agent or a label or a marker is attached. Forexample, the polypeptide, comprising the CD1d binding single-domainantibody may be linked to a pharmaceutical active agent that preferablyis delivered to a CD1d expressing cell. Another example includes acompound according to the invention that comprises a CD1d-bindingsingle-domain antibody and an antigen. Such compounds may find use in,for example, dendritic cell-based vaccines. The active agent may belinked to the compound according to the invention, preferably thepolypeptide according to the invention, allowing release of the agent onits site of delivery. Another example is wherein the compound accordingto the invention, for example, the polypeptide according to theinvention comprises a label. The label may be in the form of, forexample, a fluorescent or radioactive label, but is not limited thereto.Any kind of label that allows for detecting the presence of or thelocalization of the compound according to the invention can suitably beused within the context of the invention. In another embodiment, thecompound is a polypeptide.

However, and in addition, in another preferred embodiment of theinvention, there is provided for a compound according to any of theprevious claims wherein the compound comprises further single domainantibodies, wherein the compound comprises a label, wherein apharmaceutical active agent is linked to the compound, wherein thesingle-domain antibody is humanized, wherein the compound is abispecific or multispecific compound (bi-specificity ormulti-specificity can allow the cross-linking of two antigens), whereinthe compound is a bivalent or multivalent compound (bivalency ormulti-valency can allow antibodies to bind to multimeric antigen withgreat avidity), wherein the compound is fused to an antigen, a peptideor a nucleotide sequence, wherein the compound is a liposome, a virus,and/or wherein the compound is a nanoparticle.

Also provided is a compound as disclosed herein wherein the singledomain antibody binds to human CD1d but not to human CD1a, human CD1band/or human CD1c. In other words, within the particular use intended,the compound according to the invention comprises a single domainantibody which specifically binds to human CD1d and not human CD1a, CD1band/or CD1c. Preferable the compound according to the invention doesbind to human CD1d and not to human CD1a, CD1b and/or CD1c. The singledomain antibodies represented by SEQ ID NO 1-21 are examples of singledomain antibodies that specifically bind with human CD1d. The skilledperson knows how to determine without undue burden whether a singledomain antibody is specific for human CD1d, as can be witnessed from theExamples.

As mentioned herein, it was surprisingly found that there can beprovided for compounds comprising CD1d binding single domain antibodiesthat share high amino acid identity amongst them with respect to theCDR1, CDR2 and/or CDR3 sequences. In addition, it was found that therecan be provided for compounds, comprising the single-domain antibodiesas described herein, with different functional characteristics andfeatures, as can be witnessed from the Examples. Therefore, there isalso provided for a compound as taught herein, wherein the compound iscapable of inducing maturation of dendritic cells, preferably ofmonocyte derived dendritic cells, preferably wherein the single-domainantibody has the complementarity determining regions CDR1, CDR2 and CDR3as listed in combination for VHH2 or VHH5 in Table 1, or conservativesequence variants thereof, or wherein the single-domain antibody is VHH2or VHH5 or conservative sequence variants thereof; and/or the compoundis capable of inhibiting glycolipid, for example alpha-galactosylceramide, induced CD1d-restricted T-cell, such as invariant naturalkiller T-cell, activation, preferably wherein the single-domain antibodyhas the complementarity determining regions CDR1, CDR2 and CDR3 aslisted in combination for VHH5 or VHH24 in Table 1, or conservativesequence variants thereof or wherein the single-domain antibody is VHH5or VHH24 or conservative sequence variants thereof; and/or the compoundis capable of inducing activation of CD1d-restricted T cells, such asinvariant natural killer T-cells and/or stimulating glycolipid (e.g.alpha-galactosyl ceramide) induced activation of CD1d-restricted Tcells, such as invariant natural killer T-cell, preferably wherein thesingle-domain antibody has the complementarity determining regions CDR1,CDR2 and CDR3 as listed in combination for VHH12 in Table 1, orconservative sequence variants thereof or wherein the single-domainantibody is VHH12 or conservative sequence variants thereof; and/or thecompound is capable of inducing annexin V binding (for example, bindingof annexin V to cells that were contacted with such compound; annexin Vbinding is a marker of early apoptosis) and/or apoptosis inCD1d-expressing cells, preferably CD1d-expressing tumor, preferablywherein the single-domain antibody has the complementarity determiningregions CDR1, CDR2 and CDR3 as listed in combination for VHH3, VHH6,VHH5, or VHH19 in Table 1, or conservative sequence variants thereof, orwherein the single-domain antibody is VHH3 or VHH6 or VHH8 or VHH19 orconservative sequence variants thereof.

It was found that the single-domain antibodies VHH2 and VHH5, with theCDR's as shown in Table 1, show activity towards inducing maturation ofdendritic cells, preferably of monocyte derived dendritic cells (seeExamples), as well as cytokine production, exemplified by IL-12.Compounds comprising such single domain antibodies are useful ininducing dendritic cell maturation and cytokine production, e.g. IL-12production, in vitro or in vivo, for example in the treatment ofcancers, malaria and HIV and/or as an antimicrobial or anti-viral agent.In addition, CD1d-triggering on dendritic cells can be useful invaccination approaches, as discussed herein (see, for example, Yue etal. (2010) J Immunol. 184(1):268-76; Yue et al. (2005) Proc Natl AcadSci USA. 102(33):11811-6; Teng et al. (2009) J Immunol. 182(6):3366-71;or Teng et al. (2009) J Immunol. 183(3):1911-20)

In addition, it was found (see Examples) that there can be provided fora compound according to the invention that is capable of inhibitingglycolipid, i.e. all glycolipids that can be bound/presented by CD1d,for example, alpha-galactosyl ceramide, induced CD1d-restricted T-cell,such as invariant natural killer T-cell, activation, preferably whereinthe single-domain antibody has the complementarity determining regionsCDR1, CDR2 and CDR3 as listed for VHH5 or VHH24 in combination in Table1, or conservative sequence variants thereof. Compounds comprising suchsingle domain antibodies are useful in inhibiting glycolipid (e.g.alpha-galactosyl ceramide) induced CD1d-restricted T cell (includinginvariant natural killer T-cell) activation both in vitro or in vivo,for example in research and/or for rescue-ing iNKT (invariant NaturalKiller T-cells) cells or other CD1d restricted T cell subsets fromchronic overstimulation (see, for example, Terabe et al. (2014) CancerImmunol Immunother. 63(3):199-213).

Furthermore there is provided for a compound according to the inventionthat is capable of inducing activation of CD1d-restricted T cells,including invariant natural killer T-cells and/or stimulating glycolipid(e.g. alpha-galactosyl ceramide) induced activation of CD1d-restricted Tcells, including invariant natural killer T-cells, preferably whereinthe single-domain antibody has the complementarity determining regionsCDR1, CDR2 and CDR3 as listed in combination for VHH12 in Table 1, orconservative sequence variants thereof. Compounds comprising such singledomain antibodies are useful in inducing invariant natural killer T-cellactivation in the absence of exogenously added glycolipids and/orstimulating glycolipid (including alpha-galactosyl ceramide) inducedinvariant natural killer T-cell activation both in vitro or in vivo, forexample in the treatment of cancer. iNKT cells can exert tumorcytotoxicity via (1) direct lysis of tumor cells or via (2) productionof immunoregulatory cytokines (e.g. after interacting with DC) such asIFN-γ that trigger secondary immune effectors such as NK cells,cytotoxic T cells to exert the antitumor effect. This is reviewed e.g.in Schneiders et al. (2011) Clin Immunol. 140(2):130-41.

In addition there is provided for a compound according to the inventionthat can bind to a CD1d targeting construct allowing targeting andtargeted activation of iNKT cells at a tumor site, preferably whereinthe single-domain antibody has the complementarity determining regionsCDR1, CDR2 and CDR3 as listed in combination for VHH12 in Table 1, orconservative sequence variants thereof, preferably wherein thesingle-domain antibody is VHH12. This is useful and builds upon anapproach put forward by Stirnemann K et al. J Clin Invest. 2008 March;118(3):994-1005.

Also provided is a compound that is capable of inducing an increase inannexin V binding, which is suggestive of early apoptosis, and/orinducing apoptosis in CD1d-expressing cells, preferably CD1d-expressingtumor, preferably wherein the single-domain antibody has thecomplementarity determining regions CDR1, CDR2 and CDR3 as listed incombination for VHH3, VHH6, VHH8, or VHH19 in Table 1, or conservativesequence variants thereof. Compounds comprising such single domainantibodies are useful in inducing an increase in annexin V bindingand/or apoptosis in CD1d-expressing cells both in vitro or in vivo, forexample in the treatment of cancer. This is of use in, for example,CD1d+ malignancies where it can lead to cell death, for example inmultiple myeloma (Blood. 2009 Mar. 12; 113(11):2498-507).

Also provided is for the use of such compounds comprising single domainantibodies with the different functionalities as described above, forexample in the treatment of a condition in which such functionality isbeneficial.

In a further preferred embodiment there is provided for a compound asdescribed herein wherein the compound is a single domain antibody,preferably wherein the compound is a single domain antibody that hascomplementarity determining regions CDR1, CDR2 and CDR3 as listed incombination in Table 1, or conservative sequence variants thereof, orwherein the single domain antibody has an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 1-SEQ ID NO: 21, or conservativesequence variants thereof.

Also provided is a compound comprising an antibody, preferably asingle-domain antibody which binds to human CD1d, wherein the antibody,preferably single-domain antibody comprises complementarity determiningregions CDR1, CDR2 and CDR3 wherein CDR1, CDR2 and CDR 3 has an aminoacid sequence that has at least 80%, 90%, 95% or 100% amino acidsequence identity to the amino acid sequence of respectively CDR1, CDR2and CDR3 as shows for VHH nr 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 18, 19, 20, 21, 22, 23 or 24 as shown in Table 1. Preferably thecompound, comprising an antibody, preferably a single-domain antibodyhas an amino acid sequence that has at least 80%, 90%, 95% or 100% aminoacid sequence identity to the amino acid sequence of VHH nr 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 18, 19, 20, 21, 22, 23 or 24 as shownin Table 1, or conservative sequence variants thereof. Preferably tocompound is an antibody, preferably a single stranded antibody. Each ofthe specific antibodies shown in table 1, or antibodies comprises theCDR1, CDR2, CDR3 as shown in combination therein (i.e. per VHH number)have surprising and non-obvious properties as shown in the examples anddescription. Also provided is a nucleic acid, or vector comprising suchnucleic acid encoding for a CDR1, CDR2, and/or CDR3, antibody,single-domain antibody or compound according to the invention, asdisclosed herein.

As will be appreciated by the skilled person, the compounds as describedherein may have a wide variety of uses, including as a research tool, asa diagnostic tool, as means for delivery to a target site (expressingCD1d) of, for example, a drug, both in vitro and in vivo, in targetingtwo or more different receptors, molecules and/or antigens (e.g. whereinthe compound is bi-specific or multi-specific), both in vitro and invivo, and so on. Preferably the compound as described herein is for usein medical treatment, or for in vivo use as a diagnostic agent.Conditions that may benefit from the compound disclosed herein include,but are not limited to, cancer, HIV, malaria, asthma, allergy,autoimmune diseases, inflammatory bowel diseases andgraft-versus-host-disease (GVHD). Therefore, in another embodiment,there is provided for a pharmaceutical composition comprising a compoundaccording to the invention, for example comprising a single-domainantibody as described herein. As will be understood by the skilledperson, the pharmaceutical composition may comprise another compound inaddition to the compounds as disclosed herein, for example otherpharmaceutical active ingredients and/or excipients.

Also provided is for the use of a compound as described herein, whereinthe compound is used in vitro or wherein the compound is used in an invitro diagnostic method, for example to detect CD1d expression insamples obtained from a patient, and/or to detect cells expressing CD1d.

According to another aspect of the invention, there is provided for anucleotide sequence that encodes a compound as described herein. In thisembodiment, the compound according to the invention is a polypeptide,for example the compound is a single domain antibody, for example with asequence selected from the group consisting of SEQ ID NO: 1-21, andconservative sequence variants thereof.

The sequences as disclosed herein relate to amino acid sequences. Hence,the skilled person is well capable of providing for a nucleotidesequence encoding an amino acid sequence, as it only requires using acodon table to convert amino acid sequence into nucleotide sequence.

Such nucleotide sequence may be used to operably link it to promotersequences, polyA signals etc., to provide for a genetic construct withwhich the antibody may be expressed. Such a genetic construct comprisingthe nucleotide sequence may be comprised in a host cell. Such host cellor non-human organism comprising a nucleotide sequence according to theinvention is also provided for.

In a preferred embodiment there is provided for a nucleotide sequence asdisclosed herein, and that encodes for a compound comprising an aminoacid sequence selected from the group consisting of SEQ ID NO: 22-SEQ IDNO: 42, or conservative sequence variants thereof and/or an amino acidsequence selected from the group consisting of SEQ ID NO: 43-SEQ ID NO:63, or conservative sequence variants thereof and/or an amino acidsequence selected from the group consisting of SEQ ID NO: 64-SEQ ID NO:84, or conservative sequence variants thereof.

Also provided is for a method for preparing a compound as disclosedherein, wherein the method comprises allowing a host cell comprising anucleic acid according to the invention to express the compound; andobtaining the compound. Methods for expression and obtaining are readilyknown to the skilled person.

Finally, also provided is an antibody that comprises a CDR1 and/or CDR2and/or CDR3, preferably a CDR1 and CDR2, even more preferably a CDR1,CDR2 and CDR3, wherein the CDR1 has an amino acid sequence selected fromthe group consisting of SEQ ID NO: 22-SEQ ID NO: 42, or conservativesequence variants thereof, the CDR2 has an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 43-SEQ ID NO: 63, orconservative sequence variants thereof and CDR3 has an amino acidsequence selected from the group consisting of SEQ ID NO: 64-SEQ ID NO:84, or conservative sequence variants thereof. Preferably thecomplementarity determining regions CDR1, CDR2 and CDR 3 have an aminoacid sequence that has at least 80%, 90%, 95% or 100% amino acidsequence identity to the amino acid sequence of respectively CDR1, CDR2and CDR3 as shows for VHH nr 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 18, 19, 20, 21, 22, 23 or 24 as shown in Table 1.

The antibody may be any type of antibody, including a single domainantibody, a single chain antibody, a humanized antibody, a 4-chainantibody or any other immunoglobulin molecule. The antibody may belinked to other function or non-functional groups, for example theantibody may be a bi-specific or multi-specific antibody, and/or abi-valent or multi-valent antibody, may comprise a label of be fused toe.g. a nanoparticle, a drug, a peptide, a nucleic acid, and so on, andas disclosed herein above. The antibody may be used in treatment of a(human) patient for example, in the treatment of cancer, or may be usedto bind and detect human CD1d and/or cells expressing human CD1d.

Where the provided SEQ ID NO 22-84 in the sequence listing differs fromthe sequences shown in Table 1, the sequence shown in Table 1 prevails.

TABLE 1 VHH number, VHH reference number as used herein, and sequence ofCDR1, CDR2 and CDR3 of the various CD1d antibodies of the invention.VHH nr VHH CDR1 (SEQ CDR2 CDR3 (SEQ ID) ref ID 22-42) (SEQ ID 43-63)(SEQ ID 64-84)  1 (1) 17-1E GSSFSSYTMG AIRWSGESPYYADSVKGRLVPPGIPIERSLENMNYW  2 (2) 17-2B GRSFSSYTMG VIRWSGESPYYADSVKGRLVPPGIPIERTLESMNYW  3 (3) 17-3D GSSFSSYTMG AIRWSGESPIYADSVKGRLVPPGIPIERTLESMRYW  4 (4) 17-4C GRSFSSYTMG AIRWSGESPYYADSVKGRLVPPGIPIERTLESMKDW  5 (5) 17-7C GSSFSSYTMG GIRWSDESPIYADSVKGRLVPPGIPIPRTSESMRYW  6 (6) 17-8B GSSFSSYTMA AIRWSGESPIYADSVKGRLVPPGIPIERTLESMRYW  7 (7) 17-9C VSSFSSYTMG GIRWDDENPYYADSVKGRLVPPGIPFERTLENMRYW  8 (8) 17-10B GSSFSSYTMG AIRWDGESPIYAESVKGRLVPPGIPIERTLESMRYW  9 (9) 17-11B GRSFSSYTMG VIRWSGESPYYADSVKGRLVPPGIPIERTLESMNYW 10 (10) 19-12G GSSFSSYTMG AIRWSDESPIYAGSVKGRLVPPGIPIERTLESMRYW 11 (11) 17-13E GSSFSSYTMG AIRWSDESPYYSDSVKGRLVPPGIPIERTLENMRYS 12 (12) 18-14B GSMFSDNVMG TIRTGGSTNYADSVKGTIPVPSTPYDYW 13 (13) 19-15G GRSFSSYTMG AIRWSGESPYYADSVKGRLVPPGIPIERTLENMNYW 14 (14) 19-22H GSSFSSYTMG AIRWSGESPYYADSVKGRLVPPGIPIERTLESMNYW 18 (15) 19-21F GSSFSSYTMG AIRWSGESPIYADSVKGRLVPPGIPIERTLESMKDW 19 (16) 19-23G GSSFSSYTMT GIRWSGESPYYADSVKGRLVPPGIPIERTLESMRYW 20 (17) 19-24D GSSFSSYTMG AIRWSGESPYYGDSVKGRLVPPGIPIGRTLESMNNW 21 (18) 19-25F GSSFSSYTMG AIRWSGESPYYADSVKGRLVPPGIPIERALENMNYW 22 (19) 19-26A GSSFSSYTMG AIRWSDESPIYADSVKGRLVPPGIPIERTLESMRYW 23 (20) 19-27F GRSFSSYTMG AIRWSGESPYYADSVKGRLVPPGIPIERSLENMNYW 24 (21) 18-29C GSIFSINAMG VISSSGSTNYADSVKGHVAGFDEYNYW

TABLE 2 Sequence identity in CDR1, CDR2 and CDR3 compared to 17-1E CDR1CDR2 CDR3 17-2B  90% 94% 89% 17-3D 100% 94% 84% 17-4C  90% 100%  79%17-7C 100% 82% 73% 17-8B  90% 94% 84% 17-9C  90% 76% 84% 17-10B 100% 82%84% 17-11B  90% 94% 89% 19-12G 100% 82% 84% 17-13E 100% 88% 84% 18-14B 60% 64% 21% 19-15G  90% 100%  95% 19-22H 100% 100%  89% 19-21F 100% 94%79% 19-23G  90% 94% 84% 19-24D 100% 94% 79% 19-25F 100% 100%  95% 19-26A100% 88% 84% 19-27F  90% 100%  100%  18-29C  60% 64% 15.7% 

Examples

Immunization

Two individual llamas (Lama glama) were immunized as described (RooversR C et al. Cancer Immunol Immunother. 2007; 56:303-17). Briefly, 10⁸stable CD1d transduced C1R-cells were injected s.c. on days 0, 14, 28and 35. For phage display library construction 150 ml blood wascollected on day 43.

Selection of CD1d Specific VHH

For construction of a phage display library peripheral blood lymphocytes(PBL) were isolated from the collected 150 ml blood samples. From theisolated lymphocytes, cDNA was prepared and used as template to amplifygenes coding for the variable domains of the heavy-chain onlyantibodies. The PCR fragments were ligated into pUR8100 phagemid vectorand transformed in E. coli cells. In this way, two VHH libraries wereobtained which were subsequently expressed on phages and used forselection. For this purpose, phages from both libraries were incubatedfor 2 hours at 4° C. with CD1d transfected HeLa-cells. Cells were thenwashed and bound phages were eluted with 100 mM HCl for 7 minutes at 4°C. Removed phages were then neutralized with Tris-HCl followed byinfection into E. coli. Selected phages were then counterselected twicefor 1 hour at 4° C. using wild type C1R-cells, after which unboundphages were incubated for 1 hour with CD1d transfected C1R-cells. Boundphages were then eluted and infected to E. coli as described above.Bacteria were plated on agar plates containing 2% glucose/ampicillin togenerate single bacterial colonies coding VHH DNA. VHH DNA fromindividual clones was digested with Sfi1/BstEII digestion enzymes andcloned into plasmid pMEK219, a derivative from pHen1 (Hoogenboom H R, etal. Nucleic Acids Res 1991; 19:4133-4137). with addition of a HC-Vcassette to enable Sfi1/BstEII cloning, and a C-terminal myc- and6×HIS-tag deletion of the genIII sequence. pMEK219-VHH was transformedto E. coli TG1 bacteria.

An overnight culture was used to inoculate 2×TY medium plus 0.1% glucoseand 100 ug/ml ampicillin. When OD600 reached IPTG was added to a finalconcentration of 1 mM. Protein production was allowed for 2-5 hours.Growth of all cultures was performed at 37° C. while vigorously shakingat 200-220 rpm. Protein production was stopped by spinning cultures for15 minutes at 4° C. The bacterial pellet was resuspended in PBS andfrozen for at least 1 hour at −80° C. Bacterial suspension was thawed,slightly shaken for 1 hour at 4° C. and spun at 4500 rpm for 30 minutes.Supernatant was used to confirm binding to CD1d transfected C1R-cellsusing flowcytometry.

CD1d Specificity of Selected VHH

Confirmation of CD1d specific binding was assessed by flowcytometryusing C1R and K562 cells expressing either CD1a, CD1b, CD1c, or CD1d.Staining was performed in a 96-well plate and all incubations wereperformed in FACS buffer for 30 minutes at 4° C. For initial screeningsof binding to CD1d, cells were incubated with 25 μl supernatantcontaining anti-CD1d VHH. After washing, cells were incubated withanti-myc tag antibody clone 4A6 (Merck Millipore, MA, USA), finaldilution 1:500, washed and incubated with goat-anti-mouse F(ab)2 APC(Beckman Coulter, Fullerton, Calif., USA), final dilution 1:200. After afinal washing step, VHH binding to cells was assessed by flowcytometry(FACSFortessa, BD Biosciences). VHH showing specific binding wereselected. As a positive control the anti-CD1d 51.1 mAb (eBiosciencesInc, New Jersey, USA) was used, as negative control a nanobody specificfor azo-dye RR6 was used. Binding of the selected anti-CD1d VHH to CD1dwas confirmed after purification (see below) and sequencing of anti-CD1dVHH. For these experiments, anti-CD1d VHH and controls were tested at aconcentration of 5 μg/ml. Representative data is shown in FIG. 1.

Fingerprint Analysis and Sequencing

To select structurally different CD1d-specific VHH, DNA from selectedVHHs was amplified by colony PCR, digested with Hinf1 and subsequentlyrun on a 2% agarose gel. Based on the digestion pattern differentfamilies could be selected. Individual families were then sequenced(BaseClear B.V. Leiden, The Netherlands) to confirm unique clones.

VHH Production and Purification

Supernatants containing unique anti-CD1d VHH were produced as described.For purification, these supernatants were subsequently incubated withwashed Talon resin (Clontech, Mountain View, Calif., USA) for 1 hour atroom temperature. Talon resin was washed 3 times with PBS and once with15 mM imidazole/PBS pH 7 and eluted with 150 mM imidazole/PBS pH 7. Theeluted fraction was dialyzed twice for 24 h against PBS. Concentrationof purified VHH was determined by Nanodrop measurement (Thermo FisherScientific Inc., Wilmington, Del., USA) and purity was confirmed bycoomassie stained protein gel.

Anti-CD1d Mediated moDC Maturation

Immature monocyte derived dendritic cells (moDC) were generated asdescribed (Lameris R. et al, Methods Mol Biol, 2014; 1139: 155-65). moDCwere cultured in complete medium (RPM1-1640 containing HEPES, 10% FCS,0.05 mM beta-mercaptoethanol, (β-ME), 100 IU/mL of sodium penicillin,100 μg/mL of streptomycin sulfate, and 2.0 mM of I-glutamine) in 48-wellplates at a concentration of 6*10⁴ cells/well in the presence of 5 ng/mlrhIL-4, 500 U/ml rhGM-CSF, 1000 U/ml rhINF-γ, 25 μg/ml polymyxin B and500 nM anti-CD1d VHH or negative control VHH. LPS (200 ng/ml) was usedas a positive control. After 24 h supernatants were taken for analysisof IL-12 and 11-10 production (not shown) (using ELISA). After 72 hcells were harvested and analyzed for expression of moDC maturationmarkers (PE labelled anti-CD86 (not shown), APC labelled anti-CD83, BDBiosciences) using flowcytometry (FACS Fortessa, BD Biosciences).Representative data is shown in FIG. 2.

Inhibition of αGalCer-Induced iNKT Activation by Anti-CD1d VHH

iNKT cells were generated as described (Lameris R. et al, Methods MolBiol 2014; 1139: 155-65). 5*10⁴ CD1d-transfected HeLa cells werecultured overnight at 37° C. in a 96-well plate in DMEM, containing 10%FCS, 0.05 mM β-ME, 100 IU/mL of sodium penicillin, 100 μg/mL ofstreptomycin sulfate, 2.0 mM of 1-glutamine and 400 ng/ml α-GalCer.HeLa-CD1d cells were then washed and incubated with 500 nM anti-CD1d VHH(or negative control VHH) for 2 hours at 37° C. after which 5*10⁴resting (<25% CD25 expression) iNKT were added. After 24 h, supernatantswere harvested for detection of IFN-γ and IL-4 (using ELISA) while iNKTcells were harvested, resuspended in FACS buffer and analyzed byflow-cytometry in order to detect the induction (or inhibition) of iNKTcell activation (assessed by expression of the activation marker CD25 oniNKT cells (FACS Fortessa, BD Biosciences). See FIG. 3 and FIG. 7 forrepresentative results with at least VHH24.

Induction of iNKT Cell Activation by Anti-CD1d VHH

iNKT cells were generated as described (Lameris R. et al, Methods MolBiol 2014; 1139: 155-65). 5*10⁴ CD1d-transfected HeLa cells,CD1d-transfected C1R cells and CD1d-transfected MM.1s cells werecultured overnight at 37° C. in a 96-well plate in DMEM, containing 10%FCS, 0.05 mM β-ME, 100 IU/mL of sodium penicillin, 100 μg/mL ofstreptomycin sulfate, 2.0 mM of 1-glutamine in the presence or absenceof 100 ng/ml α-GalCer or vehicle control. CD1d-transfected cells, loadedwith α-GalCer or vehicle control, were then washed and incubated with500 nM anti-CD1d VHH (or negative control VHH) for 2 hours at 37° C.after which 5*10⁴ resting (<25% CD25 expression) iNKT cells were added.After 24 h, supernatants were harvested for detection of IFN-γ and IL-4(using ELISA) while iNKT cells were harvested, resuspended in FACSbuffer and analyzed by flow-cytometry in order to assess the inductionof iNKT cell activation (assessed by expression of the activation markerCD25 on iNKT cells (FACS Fortessa, BD Biosciences). See FIG. 4 and FIG.8 (concentration dependency) for representative results; showing datafor VHH12.

Analysis of Annexin V Binding Induced by Anti-CD1d VHH

CD1d-C1R and CD1d-MM.1s (as well as untransfected C1R and MM.1s celllines as negative controls) were cultured at 37° C. in a 48-well plateat 1*10⁵ cells per well and incubated with 500 nM anti-CD1d VHH,negative control VHH, or anti-CD1d 51.1 mAb (as positive control). After24 h, cells were stained with annexin V and propidium iodide (P1)according to manufacturers protocol (VPS Diagnostics, Hoeven, theNetherlands) and analyzed by flow cytometry (FACS Fortessa, BDBiosciences). Experimental results are shown in FIG. 5.

Induction of iNKT Cell Activation by Platebound CD1d and Anti-CD1d VHH

iNKT cells were generated as described (Lameris R. et al, Methods MolBiol 2014; 1139: 155-65). α-GalCer (1 mM) or vehicle control (100% DMSO)were heated for 2 minutes at 80° C., sonicated for 5 minutes andsubsequently diluted in sterile, warm (37° C.) 0.1% triton-X to aconcentration of 100 μM. Next 6 μM of a bispecific construct consistingof an anti-EGFR VHH fused to β2m-human CD1d was added in a 1:1 ratio.Final concentrations of α-GalCer and β2m-CD1d-anti-EGFR construct were50 μM and 3 μM respectively. Vehicle and α-GalCer where incubatedovernight at room temperature while shaking. 96-well plates were coatedwith anti-flag mAb (Sigma, clone M2; 1:1000) and incubated overnight at4° C. The next day anti-flag coated plates were washed trice with PBSand incubated with α-GalCer or vehicle loaded construct diluted in PBS(construct concentration 0.5 μM) for 2 hours, while shaking at roomtemperature. After washing with PBS, coated plates were incubated with250 nM anti-CD1d VHH for 2 hours at 37° C. after which 1*10⁵ resting(<25% CD25 expression) iNKT cells were added. After 24 h iNKT cells wereharvested, resuspended in FACS buffer and analyzed by flow-cytometry inorder to assess the induction of iNKT cell activation (assessed byexpression of the activation marker CD25 on iNKT cells (FACS Fortessa,BD Biosciences). Results are presented in FIG. 6.

VHH12

In addition to the data shown above, additional experiments wereperformed using VHH12. The results of the experiments are shown in FIG.9, FIG. 10 and FIG. 11. FIG. 9 shows induction of iNKT celldegranulation (left) and cytotoxicity against CD1d+ tumor cells line(right). FIG. 10 shows induction of iNKT cell cytotoxicity against CD1d+primary multiple myeloma cells. FIG. 11 shows induction of iNKT cellcytokine production by anti-CD1d VHH12. For detection of cytokineproduction HeLa-CD1d cells were pulsed with vehicle control, OCH (asphingosine truncated derivative of alpha-galactosylceramide (alpha-GC);glycolipid reported to induce Th2-cytokine production in iNKT cells) orαGC, incubated with anti-CD1d VHH and controls and co-cultured with iNKTfor 24 h after which supernatants were analyzed (by Cytometric BeadAssay; CBA). N=4; *p<0.05; ****p<0.0001. The anti-CD1d VHH shown isVHH12.

Results

Representative results of the various experiments is shown in theFigures and the accompanying legends; additional experimental data isdiscussed above in the context of the current invention.

The invention claimed is:
 1. A compound comprising at least onesingle-domain antibody which binds to human CD1d, wherein thesingle-domain antibody comprises complementarity determining regionsCDR1, CDR2 and CDR3, wherein CDR1 comprises the amino acid sequence setforth in SEQ ID NO:33, CDR2 comprises the amino acid sequence set forthin SEQ ID NO:54 and CDR3 comprises the amino acid sequence set forth inSEQ ID NO:
 75. 2. A compound according to claim 1, wherein the compoundcomprises the amino acid sequence set forth in SEQ ID NO:12, or aconservative sequence variant thereof.
 3. A compound according to claim1, wherein the compound comprises a label, wherein an pharmaceuticalactive agent is linked to the compound, wherein the compound is abivalent or multivalent compound, wherein the compound is fused to anantigen, a peptide or a nucleotide sequence, wherein the compound is aliposome, wherein the compound is a virus, and/or wherein the compoundis a nanoparticle.
 4. A compound according to claim 1, wherein thecompound binds to human CD1d but not to human CD1a, human CD1b and/orhuman CD1c.
 5. A compound according to claim 1, wherein the compound iscapable of inducing activation of CD1d-restricted T cells, includinginvariant natural killer T-cells, and/or stimulating alpha-galactosylceramide, induced activation of CD1d-restricted T cells, includinginvariant natural killer T-cells.
 6. A compound according to claim 1,wherein the compound is a single domain antibody.
 7. A compoundaccording to claim 1, wherein the single domain antibody is a humanizedsingle domain antibody.
 8. A compound according to claim 1, for use inmedical treatment, or for in vivo use as a diagnostic agent.
 9. Apharmaceutical composition comprising a compound according to claim 1.10. A nucleotide sequence that encodes a compound according to claim 1.11. An isolated host cell or non-human organism comprising a nucleotidesequence encoding a compound according to claim
 1. 12. The host cellaccording to claim 11 that produces said compound or part thereof.
 13. Amethod for preparing a compound comprising at least one single-domainantibody which binds to human CD1d, wherein the method comprisesculturing the host cell of claim 12 to express the compound or partthereof; and obtaining the compound.
 14. An antibody that comprises aCDR1, CDR2 and CDR3, wherein the CDR1 has the amino acid sequence setforth in SEQ ID NO:33, the CDR2 has the amino acid sequence set forth inSEQ ID NO:54 and CDR3 has the amino acid sequence set forth in SEQ IDNO:75.
 15. A compound according to claim 1, wherein the compoundcomprises the amino acid sequence set forth in SEQ ID NO:
 12. 16. Acompound according to claim 1, wherein the compound further comprises anadditional single domain antibody.
 17. A compound according claim 1,wherein the compound is a bispecific or multispecific compound.
 18. Acompound according claim 1, wherein the compound is linked to anotherantibody.